In the previous four years of this PEGT, we carried out AAV-mediated gene delivery of FVIII cDNA to hemophilia A mice and dogs. Recently, we found that use of the new AAV capsid serotypes, AAV8 and AAV9, to deliver canine FVIII heavy and light chain sequences in either two separate vectors or a single vector led to complete long-term correction of hemophilia A mice. This result was obtained with either intraportal or intravenous delivery. However, results from intraportal delivery of canine heavy and light chain cDNAs to hemophilia A dogs in an AAV8 vector have been disappointing. One dog in three has had partial correction of 4-8% activity for 18 months, while the other two had only 1% FVIII activity. Since the best animal model and best developmental stage for FVIII gene therapy are unknown, we now have decided to turn to a non-human primate, the rhesus macaque, and to carry out gene delivery in the fetus and neonate. In Specific Aim 1, we develop the tools for monkey studies, cloning human FVIII cDNA in two separate constructs and developing a human-specific FVIII antibody. In Specific Aim 2, we study the effectiveness of three different AAV serotypes, AAV8, AAV9, and the best available hybrid AAV vector, at two different doses of vector. In this Aim, two fetal monkeys will be evaluated for each of the six conditions with all fetuses undergoing intrahepatic delivery of vector in late first trimester. In Specific Aim 3, we will validate the optimal conditions for effective FVIII gene delivery discovered in Specific Aim 2 using neonatal monkeys and neonatal hemophilia A dogs. Thus, in this renewal we will find conditions for safe, long term FVIII expression in early development as a model for a useful treatment option for hemophilia A.